1. Field of the Invention
This invention is related in general to electrodes for electrophysiological testing of biological membranes and, in particular, to a cell-delivery unit that is especially suitable for massive parallel testing of cells. The invention includes a reusable cell-delivery component that is separable from the patch-clamp component of the electrode assembly, a method for automatically aligning the two components rapidly and reliably using electric-field probing, and a method for positioning test cells accurately on the patch aperture using dielectrophoresis.
2. Description of the Related Art
In conventional patch clamping used to conduct electrophysiological tests on membranes, a micropipette with a heat-polished tip of about 1 to 5 μm in diameter is physically sealed to a “patch” on the membrane. The electrical activity in the membrane is then assessed by measuring current or voltage changes produced in response to exposure to various test stimuli, such as voltage step changes, current injection, the application of compounds, or mechanical stimulation. Typically, the membrane potential is controlled while the current flowing through the membrane is measured to detect ion-channel activity that corresponds to changes in the membrane's conductance.
For the most part, patch clamping is used either in a whole-cell or a single-channel mode of operation. In whole-cell patch clamping, the membrane at the tip of the pipette is ruptured to produce electrical continuity between the electrolyte in the pipette and the interior of the cell. Thus, total membrane current or voltage is measured. In single-channel patch clamping, the integrity of the membrane at the tip of the pipette is preserved. Accordingly, the recorded current is only the current flowing through the patch of the membrane enclosed by the tip of the pipette. Since this area is very small, there is a good chance that only one or a small number of ion channels may be in the membrane patch, and individual ion-channel currents may be recorded.
In both types of patch-clamp techniques, when the tip of the pipette is pressed against the cell membrane, the interior of the pipette is isolated from the extracellular solution by the seal that is formed between the tip of the pipette and the membrane. If the electrical resistance of the seal is very high (in the order of several hundred mega-ohm to several giga-ohms), no current can leak across the seal and good measurements are obtained. Thus, any leakage of current through the seal is undesirable and the creation of a high-resistance seal (in the order of giga-ohms) is crucial for good results.
New planar patch-clamp electrodes have been developed in the art based on a seal formed by a test cell's membrane and an aperture in a nonconductive partition separating the extracellular carrier solution from an intracellular electrolyte. Typically, these systems comprise multiple perfusion chambers where the partition separates a top (extracellular) compartment, where the test cells are suspended in an extracellular solution, from a bottom (intracellular) compartment containing an electrode and an intracellular solution. The plate configuration of the partition allows the manufacture of a disposable tray with multiple perfusion chambers for parallel testing of large numbers of cells. For instance, International Application No. PCT/IB98/01150 describes a perforated partition with multiple holes for forming a plurality of patch-clamp seals between intra and extracellular compartments.
Patch clamp requires placing a test cell onto the aperture connecting the two compartments of each chamber. Thus, a planar partition between intracellular and extracellular compartments provides a patch-clamp aperture (often also called “pore” in the art) in each perfusion chamber. The dimension of a typical aperture is about 2 micron and the precise positioning of a test cell over the aperture is a critical factor for the speed of operation of a patch-clamp system. In conventional (manual) patch clamp, the cell is brought into contact with the aperture (which in that case is the pore in the tip of a patch pipette) by manipulating the pipette under microscopic control by a skilled operator. Obviously, high-throughput electrophysiology cannot be accomplished by this process and requires an approach suitable for automation.
High-throughput systems described in the art incorporate the cell-positioning compartment and the patch-clamp compartment into a single structure, typically implemented in “chip” format. One such system is described, for example, in U.S. Patent Application No. 2002/0182627. For successful operation, the test cell needs to be placed in the immediate vicinity of the patch pore in each perfusion chamber, preferably within a few microns from the pore, with subsequent application of solution suction through the pore to bring the cell directly into the pore. In the case of a planar patch, the pore is an aperture in an essentially flat, electrically insulating substrate separating the two compartments. Several ways have bee proposed for accomplishing this initial positioning of the test cells in an automated system, such as using microfluidics to create solution flow in the vicinity of the pore that brings the cell to the pore (disclosed in commonly owned U.S. Ser. No. 09/973,388, herein incorporated by reference), and using voltage applied across the pore to create electric fields in the solution which attract the cell to the pore by dielectrophoretic forces (as described, for example, in International Application No. PCT/IB98/01150 and U.S. Publication No. 2002/0144905). In practice, microfluidics flow requires complicated and expensive channels in the vicinity of the pore.
Thus, in addition to the ability to form a high-resistance seal, high-throughput electrophysiological testing requires a reliable and practical approach to the challenge of accomplishing the initial positioning of test cells in the vicinity of the patch-clamp apertures. Furthermore, inasmuch as disposable patch-clamp partitions need to be replaced between tests, the single-structure construction of high-throughput electrophysiological perfusion apparatus adopted in the art greatly contributes to the high cost of operating these systems. This invention provides a different approach that substantially improves these problems.